Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks.

Author(s): Guyard A, Boyez A, Pujals A, Robe C, Tran Van Nhieu J, Allory Y, Moroch J, Georges O, Fournet JC, Zafrani ES, Leroy K

Publication: Virchows Arch, 2017, Vol. , Page

PubMed ID: 28812131 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   TThe purpose of this study was to investigate the effect of FFPE block storage on DNA yield, amplificability, and integrity as well as on mutation detection and to investigate if tissue-type is a confounding factor. Colon (21), lung (8), and urothelial tract (17) cancer specimens were fixed in neutral buffered 4% formaldehyde for an unspecified duration and paraffin embedded. DNA was extracted before and after 4-6 years of storage without temperature or humidity control for 4-6 years. Sections were H&E-stained and DNA was extracted using the BioRobot EZ1 with the EZ1 DNA Tissue Kit. While DNA was extracted 4-6 years apart, the same macrodissection area, number and thickness of FFPE sections, and elution volume were used for both extractions and DNA was subsequently stored at -20˚C. DNA yield was determined by fluorometry. The amount of amplifiable DNA was determined by amplification of a 137 bp fragment of KRAS exon 2. The maximum length of amplifiable DNA was determined using theBIOMED2 assay which amplifies 100, 200, 300, 400, 500, and 600 bp amplicons and assessed by electrophoresis. Additionally, a multiplex real-time PCR assay compared amplification of 150 and 300 bp fragments with a 75 bp amplicon. Mutations were detected by NGS using the Ion AmpliSeq Colon and Lung Cancer Panel and by high resolution melting analysis of KRAS exon 2.

   Summary of Findings:

   The DNA yield was decreased for 42 of the 46 blocks after storage (mean of 71 ng/µL versus 32 ng/µL, P<0.0001). However, the decrease in DNA yield with storage was dependent on tissue-type with average decreases of 66% (P<0.001), 38% (P<0.01), and 28% (P<0.05) found for colon, urothelial tract, and lung, respectively (P<0.024). Similarly, the amount of amplifiable DNA decreased from an average of 56% in fresh blocks to 15% for stored blocks (P<0.0001), with decreases from 56% to 15% noted for colon specimens (P<0.0001), 75% to 18% for urothelial specimens (P<0.01), and 26% to 4% for lung specimens (P<0.01). The median percentage of amplifiable DNA that was still amplifiable after storage was comparable among tissue types (9% for colon, 13% for urothelial tract, and 11% for lung). While products of up to 400-600 bp were amplifiable in DNA from fresh blocks, only 200-300 bp fragments were amplifiable in DNA from stored blocks. The ratio of amplified products from fresh blocks of 150 bp and 300 bp relative to 75 bp decreased from 58% and 31%, respectively and decreased from stored blocks to 16% and 2%, respectively (P<0.000, both).

   High resolution melting curves for KRAS exon 2 were comparable in DNA from stored and fresh blocks, regardless of mutational status. However, NGS sequencing of 12 specimens (four of each tissue type) found the mean library yield decreased after block storage by 3.3 fold (0.7 to 17.6-fold, P=0.0108) and that block storage led to a 4.5-fold increase in mutation detection (P=0.006). The authors attribute the increased mutation detection to an increase in C>T and G>A artifacts.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Other
   • Tissue - Lung

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	PCR
   DNA	SNP assay
   DNA	Real-time qPCR
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 years 4-6 years
   Biospecimen Acquisition	Biospecimen location	Colon Lung Urothelial tract
   SNP assay Specific	Technology platform	High resolution melting Next generation sequencing
   Real-time qPCR Specific	Length of gene fragment	75 bp 150 bp 300 bp
   PCR Specific	Length of gene fragment	100 bp 200 bp 300 bp 400 bp 500 bp 600 bp

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